Sierra et al. (Anal. Chem. 69 (1997), 1471-1476) describe a determination of blood glucose based on the intrinsic fluorescence of glucose oxidase. In this method too, the enzyme is employed together with its coenzyme FAD in catalytic amounts, with redox equivalents being transferred to oxygen as mediator.
Narayanaswamy et al. (Analytical Letters 21 (7) (1988), 1165-1175) describe a fluorescence measurement with glucose dehydrogenase and NAD for glucose determination. The enzyme is in this case employed in catalytic, i.e. non-stoichiometric, amounts. The fluorescence measurement detects the free NADH in the solution.
It is possible through the electrochemically active substances (mediators) required for the prior art detection systems to detect the analytes to be determined only indirectly, i.e. via a plurality of chemical reactions. For this purpose, a complicated adjustment of the concentrations of the substances involved to optimize the reaction rate is often necessary. There is moreover the risk that the required electrochemically active substances are unstable on prolonged storage.
The mediators often also have to be employed in large excess relative to the enzyme-coenzyme system. The coenzyme has a high reactivity, so that the enzymic activity declines markedly on decomposition of the mediator, even in small amounts, e.g. <1% or on exposure to foreign substances, e.g. volatilization of the substances from packaging materials. This may lead to false signals in the determination of the analyte. Yet a further disadvantage is that the determination times for the analytes are normally in the region of at least a few seconds, for example for glucose in the region of >4 s, and the required sample volumes are large, e.g. >0.5 μl.
The object on which the present invention was based is at least partly to avoid the described disadvantages of the prior art. It was particularly intended to provide a non-sensitive and rapid method for the enzymatic detection of analytes, which leads to reliable measurement results even in the absence of mediators or/and indicators.
This object is achieved by using an enzyme-coenzyme complex as stoichiometric reactant instead of, as usual, as catalyst. Detection of the analyte requires only a single reaction step and is therefore extremely fast. The use of mediators and indicators, associated with the employment of complex reaction mixtures, with low stability and high susceptibility to interference, is no longer necessary.